Extended storage of reduced glutathione solutions

ABSTRACT

A method of storing solutions of reduced glutathione for extended periods of time, by dissolving reduced glutathione in an aqueous medium having a pH of between 5.0 and 8.0 to produce a reduced glutathione solution; reducing the temperature of the reduced glutathione solution to a predetermined temperature which is sufficiently low to prevent oxidative dimerization of glutathione without freezing the aqueous medium; and storing the reduced glutathione solution at the predetermined temperature.

This application is a continuation of co-pending U.S. application Ser.No. 09/988,253, filed on Nov. 19, 2001, herein incorporated byreference.

This application claims the benefit of U.S. Provisional Application No.60/275,139 entitled “EXTENDED STORAGE OF REDUCED GLUTATHIONE SOLUTIONS,”filed on Mar. 13, 2001.

FIELD OF THE INVENTION

The invention relates to a method of storing reduced glutathione forextended periods of time. Normally, when a solution of reducedglutathione is stored, the percentage of biologically active glutathionewhich is present in solution undergoes a significant reduction overtime. This is due to degradation of glutathione in solution. The currentinvention provides a method of storing reduced glutathione in solutionfor an extended period of time with little or no change in theconcentration of the active ingredient.

BACKGROUND ART

Glutathione, a tripeptide containing a free thiol group, is an importantantioxidant. In preliminary research, dietary glutathione intake fromfruit and raw vegetables has been associated with protection againstsome forms of cancer. Glutathione has also inhibited cancer in test tubeand animal studies. In preliminary research, higher glutathione levelshave also been associated with good health in older adults. In fact,glutathione levels appear to show a very strong correlation with lifeexpectancy. The reduced glutathione (G-SH) molecule consists of threeamino acids—glutamic acid, cysteine, and glycine—covalently joinedend-to-end. The sulfhydryl group, which gives the molecule itselectron-donating character, comes from the cysteine residue.Glutathione is present inside cells mainly in its reduced form.

Studies using intravenous or intramuscular glutathione have found it tobe useful for preventing clot formation during operations; reducing theside effects and increasing the efficacy of chemotherapy drugs(particularly cisplatin in women with ovarian cancer); treatingParkinson's disease; reducing blood pressure in diabetics with highblood pressure; and increasing sperm counts in men with low spermcounts. Whether oral preparations are also effective is unknown at thistime. A small study in eight patients with liver cancer using oralglutathione showed modest effects in women but not in men when given ina daily amount of 5,000 mg.

In the healthy cell, the concentration of oxidized glutathione (G-SS-G)is normally less than 10% of the total glutathione concentration. Theratio of reduced glutathione to oxidized glutathione appears to be animportant indicator of the cell's health, and of its ability to resistoxidative stress. A reduction of reduced glutathione in a cell cantrigger suicide of the cell by a process known as apoptosis.

Reduced glutathione (GSH) works as a potent anti-oxidant in thetreatment of Parkinson's disease. According to research by Dr. DavidPerlmutter on alterations in glutathione levels, there seems to be botha clinical and neuropathological difference in Parkinson's diseasepatients treated with IV glutathione versus control groups. Dr.Perlmutter measured both GSH levels and oxidized glutathione (GSSG)levels in the brains of both Parkinson's patients and in the brains ofpatients in control groups. Glutathione levels were reducedapproximately 40% and oxidized glutathione was increased approximately29% in the patients with Parkinson's disease. This altered GSH/GSSGratio in the brain may indicate that oxidative stress is a factor inbrain cell death in Parkinson's disease. Therefore, treatment ofParkinson's disease with pure reduced glutathione may help to delay theprogression of the disease.

Treatment of patients with lung diseases such as pulmonary fibrosis andemphysema with reduced glutathione is also useful. Reduced glutathioneacts as an antioxidant, preventing oxidants in pollution and cigarettesmoke from causing further damage to the lungs.

Unfortunately, reduced glutathione is not stable when subjected tolong-term storage. The thiol group on the glutathione undergoes gradualoxidation to a disulfide, as shown:

This reaction is catalyzed by the presence of molecular oxygen or ofcertain metal ions, such as Fe⁺³, but it will proceed in aqueoussolution in the absence of catalytic agents. As this reaction proceeds,there is a gradual reduction in the efficacy of the reduced glutathionesolution against Parkinson's. In fact, if greater than 10% of the totalglutathione in the reduced glutathione solution has undergone oxidation,the treatment may conceivably increase oxidative stress in the brain,exacerbating Parkinson's disease.

Thus, there is a long-felt need in the art for a method of storingaqueous solutions of reduced glutathione without allowing oxidation ofthe glutathione to a disulfide.

SUMMARY OF THE INVENTION

The current invention is directed toward extended storage of reducedglutathione in an aqueous medium having a pH of between 5.0 and 8.0,preferably between 6.2 and 7.8. The solution of reduced glutathione hasa defined initial concentration. Even in the absence of oxidationcatalysts such as molecular oxygen and/or metal ions, the concentrationof reduced glutathione frequently undergoes a gradual reduction ofbetween 10% and 15% of the initial concentration per month. It has beenunexpectedly found that storing the reduced glutathione solution at areduced temperature, which is above the freezing point of the solutionand below 15° C., maintains the rate of reduced glutathione decay atbetween 0% and 5% of the defined initial concentration per month.

Thus, this invention provides a method for extended storage of reducedglutathione by dissolving a predetermined quantity of reducedglutathione in an aqueous medium having a pH of between 5.0 and 8.0 toproduce a reduced glutathione solution having a predetermined initialconcentration; reducing the temperature of the reduced glutathionesolution to a predetermined temperature which is sufficiently low toprevent oxidative dimerization of glutathione without freezing theaqueous medium; and storing the reduced glutathione solution at thepredetermined temperature.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the variation in the concentration of glutathione in anaqueous glutathione solution as a function of time, recorded at roomtemperature.

FIG. 2 shows the variation in the concentration of glutathione in anaqueous glutathione solution as a function of time, recorded underconditions of refrigeration.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As previously noted, reduced glutathione (G-SH) undergoes an oxidativedimerization reaction, according to the chemical equation:

When the ratio of the actual concentration of reduced glutathione [G-SH]at time t (recorded in Table 1) to the initial concentration of reducedglutathione [G-SH]_(o) in a solution which is free of oxidationcatalysts, such as metal ions and/or molecular oxygen, is plotted as afunction of time (see FIG. 1), the ratio [G-SH]/[G-SH]_(o) decreaseswith time in a linear fashion with a temperature-dependent slope K. Itis important that solutions of reduced glutathione be used before theconcentration of the oxidized disulfide of glutathione builds up to asignificant extent, particularly when treating Parkinson's disease.Otherwise, administration of a reduced glutathione solution will fail tocompensate for the altered GSH/GSSG ratio in the brain of Parkinson'spatients. Preferably, the reduced glutathione solution is administeredwhile the concentration of reduced glutathione is still at least 90% ofthe initial reduced glutathione concentration. As a result, reducedglutathione cannot be safely stored in aqueous solution at roomtemperature for longer than three weeks, even in the absence ofoxidation catalysts (see Table 1).

The rate at which the concentration of reduced glutathione in solutiondeclines is dependent in large part on the rate constant k forglutathione oxidation:

The rate constant for a reaction is well known to be temperaturedependent, according to the Arrhenious equation:k=A exp (−E _(a) /RT)

where E_(a) is the activation energy of the reaction, R is the universalgas constant, T is temperature, and A is a temperature-independentpreexponential factor. TABLE 1 Effect of Long-Term Storage at 25° C. onReduced Glutathione Concentration [G-SH]/[G-SH]₀ Time (days) 0.94 1 0.992 0.99 3 0.98 7 0.95 14 0.90 21 0.89 28 0.78 56 0.68 74

Thus, for most reactions, the rate constant k increases with increasingtemperature. Therefore, it should be possible to slow the rate ofglutathione oxidative dimerization down by decreasing the temperature. Atypical reaction rate having an activation energy of roughly 50 kJ mol⁻¹changes by a factor of two for a 10° temperature change. Therefore, itcan be anticipated that the rate of reaction for glutathionedimerization can be decreased by a factor of four by lowering thetemperature of the solution from room temperature by about 20°.

However, it has unexpectedly been found that the rate of glutathionedimerization is not simply reduced by lowering the solution temperatureby 20° from room temperature. Rather, the dimerization of glutathione isessentially stopped (see Table 2), allowing the storage of the reducedglutathione solution for periods of time of significantly greater thanfour months. TABLE 2 Effect of Long-Term Storage at 5° C. on ReducedGlutathione Concentration [G-SH]/[G-SH]₀ Time (days) 1.02 1 1.01 2 1.013 1.02 7 1.00 14 0.99 21 1.02 28 1.00 56 1.00 84 0.98 112

Additionally, dramatic reductions in the rate of oxidative dimerizationof glutathione can be achieved through storage of a reduced glutathionesolution at a temperature which is above the freezing point of theaqueous medium and below 15° C. Under these conditions, the rate ofglutathione dimerization is dramatically reduced, with the concentrationof reduced glutathione decreasing at a rate of between 0% and 5% of theinitial reduced glutathione concentration per month.

Thus, this invention provides a method for extended storage of reducedglutathione in solution, by dissolving a predetermined quantity ofreduced glutathione in an aqueous medium to produce a reducedglutathione solution having a predetermined initial concentration;reducing the temperature of the reduced glutathione solution to apredetermined temperature which is sufficiently low to slow the rate ofoxidative dimerization of glutathione without freezing the aqueousmedium; and storing the reduced glutathione solution at thepredetermined temperature. The aqueous medium is preferably a solutionof reduced glutathione at a concentration of between 10 mg/ml and 400mg/ml having a pH of between 5.0 and 8.0. The solution may additionallycomprise a buffer, such as octylammonium orthophosphate, to control thepH of the solution. It is preferred that the solution be stored under anon-oxidizing atmosphere which is free of molecular oxygen (i.e.,nitrogen, argon, or a vacuum), and that the solution be prepared usingdistilled water which is free of metallic ions. The solution may bestored in single-dose or multi-dose ampoules or vials. If the solutionis stored in multi-dose ampoules or vials, it is preferred that theampoules or vials should be sealed with an elastomeric septum. Thesolution can then be withdrawn by syringe through the septum, whileminimizing access of atmospheric oxygen to the reduced glutathionesolution.

Additionally, this invention provides a method for treating a patientwith Parkinson's disease with reduced glutathione while minimizing therisk of oxidative stress on the brain. This is done by dissolving apredetermined quantity of reduced glutathione in an aqueous mediumhaving a pH of between 5:0 and 8.0, preferably between 6.2 and 7.8, toproduce a reduced glutathione solution; storing the reduced glutathionesolution at the predetermined temperature; and administering the reducedglutathione to the patient with Parkinson's disease. This eliminatesfurther deleterious effect on the health of brain cells from increasesin the level of oxidized glutathione relative to reduced glutathione.The glutathione solution may be administered intravenously, byintramuscular injection, by subcutaneous injection, or orally. Theglutathione solution may also be administered orally, in either a liquidform or as the contents of a liquid-filled capsule. If desired, agar orgelatin may be added to the aqueous contents of the capsule to provideadditional viscosity to the solution. The shell of the capsule degradesin the small intestine, releasing the glutathione. It is also possibleto add sufficient agar or gelatin to the solution of reduced glutathioneto convert the reduced glutathione to a self-sustaining gel. This gelmay then be swallowed orally, or inserted rectally and used as asuppository. Alternatively, in one embodiment, the reduced glutathionesolution may be stored under an inert atmosphere in a spray bottlehaving a sealed opening. The opening of the spray bottle may then beopened to the atmosphere with a pin or similar implement, and thecontents of the spray bottle may then be sprayed into the nasal or oralcavity. The glutathione may then be transported across the patient'smucous membranes into the bloodstream.

Finally, this invention provides a method for treating a patient withemphysema with reduced glutathione. This is done by dissolving apredetermined quantity of reduced glutathione in an aqueous mediumhaving a pH of between 5.0 and 8.0, preferably between 6.2 and 7.8, toproduce a reduced glutathione solution; storing the reduced glutathionesolution at the predetermined temperature; and administering the reducedglutathione to the patient with emphysema. The glutathione solution maybe administered by any of the methods described above for treatment ofParkinson's; however, it is preferred to provide the reduced glutathionesolution under an inert atmosphere in a spray bottle having a sealedopening. The opening of the spray bottle may then be opened to theatmosphere, and the contents of the spray bottle may then be sprayedinto the nasal cavity. This provides the most rapid access of reducedglutathione to the respiratory surfaces.

COMPARATIVE EXAMPLE

A reduced glutathione solution containing reduced glutathione at aninitial concentration of 180 mg/ml and octylammonium orthophosphate at aconcentration of 0.005M was prepared. The solution had a pH of 6.4. Thissolution was stored at room temperature (25° C.) for 74 days. Theconcentration of reduced glutathione exhibited a decrease over time,with a steady rate of decay of between 10% and 12% of the initialreduced glutathione concentration per month (FIG. 1). Within threeweeks, the concentration of reduced glutathione had decreased to 90% ofits initial value (Table 1). This decrease is sufficient to render thesolution of questionable value for the treatment of Parkinson's disease.

EXAMPLE

A reduced glutathione solution containing reduced glutathione at aninitial concentration of 189 mg/ml and octylammonium orthophosphate at aconcentration of 0.005M was prepared. The solution had a pH of 6.4. Thissolution was stored at 5° C., and the concentration of reducedglutathione was monitored for four months (Table 2). Within experimentalerror, the concentration of reduced glutathione exhibited no decreaseover time (FIG. 2).

1-8. (canceled) 9) A method for extended storage of reduced glutathionein solution, comprising the steps of: a) dissolving a predeterminedquantity of reduced glutathione in an aqueous medium free of metal ionshaving a pH of between 5.0 and 8.0 to produce a reduced glutathionesolution having a predetermined initial concentration [A]_(o); b)reducing the temperature of the reduced glutathione solution to apredetermined-temperature which is sufficiently low to prevent oxidativedimerization of glutathione without freezing the aqueous medium; and c)storing the reduced glutathione solution at the predeterminedtemperature. 10-11. (canceled) 12) A method for treating a patient witha disease selected from the group consisting of Parkinson's disease andemphysema with reduced glutathione, comprising the steps of: a)dissolving a predetermined quantity of reduced glutathione in an aqueousmedium free of metal ions having a pH of between 5.0 and 8.0 to producea reduced glutathione solution having a predetermined initialconcentration [A]_(o); b) reducing the temperature of the reducedglutathione solution to a predetermined temperature which issufficiently low to prevent oxidative dimerization of glutathionewithout freezing the aqueous medium; c) storing the reduced glutathionesolution at the predetermined temperature; and d) administering thereduced glutathione to the patient with said disease. 13) A methodaccording to claim 10, wherein the disease is Parkinson's disease. 14) Amethod according to claim 10, wherein the disease is emphysema. 15) Amethod for preparing a reduced glutathione composition, comprising thesteps of: a) dissolving a predetermined quantity of reduced glutathionein an aqueous medium free of metal ions having a pH of between 5.0 and8.0 to produce a reduced glutathione solution having a predeterminedinitial concentration [A]_(o), wherein, when the ratio [A]/[A]_(o),where [A] is the actual glutathione concentration at time t, is plottedas a function of time t, the concentration of reduced glutathionedecreases over time in a linear fashion with a temperature-dependentslope; and b) storing the reduced glutathione solution at apredetermined temperature which is above the freezing point of thesolution and below 15° C., so as to maintain the rate of reducedglutathione decay at between 0% and 5% of the predetermined initialconcentration per month. 16) The method of claim 12, additionallycomprising a step of reducing the temperature of the aqueous medium tosaid predetermined temperature subsequent to dissolution of reducedglutathione in said aqueous medium. 17) A method for preparing a reducedglutathione composition, comprising the steps of: a) dissolving apredetermined quantity of reduced glutathione in an aqueous medium freeof metal ions having a pH of between 5.0 and 8.0 to produce a reducedglutathione solution having a predetermined initial concentration,wherein the concentration of reduced glutathione decays over time in alinear fashion; b) storing the reduced glutathione solution at apredetermined temperature which is above the freezing point of thesolution and below 15° C., so as to maintain the rate of reducedglutathione decay at between 0% and 5% of the predetermined initialconcentration per month. 18) A method for treating a patient with adisease selected from the group consisting of Parkinson's disease andemphysema with reduced glutathione, comprising the step of: a)administering a reduced glutathione composition to the patient, wherethe reduced glutathione composition has been prepared according to themethod of claim
 12. 19) A method according to claim 18, wherein thedisease is Parkinson's disease. 20) A method according to claim 18,wherein the disease is emphysema. 21) A reduced glutathione composition,said composition being prepared by a process comprising the steps of: a)dissolving a predetermined quantity of reduced glutathione in an aqueousmedium free of metal ions having a pH of between 5.0 and 8.0 to producea reduced glutathione solution having a predetermined initialconcentration, wherein the concentration of reduced glutathione decaysover time in a linear fashion; b) storing the reduced glutathionesolution at a predetermined temperature which is above the freezingpoint of the solution and below 15° C., so as to maintain the rate ofreduced glutathione decay at between 0% and 5% of the predeterminedinitial concentration per month; wherein said reduced glutathionesolution is effective in the treatment of a patient according to themethod of claim
 10. 22) A method for preparing a reduced glutathionecomposition, comprising the steps of: a) dissolving a predeterminedquantity of reduced glutathione in an aqueous medium having a pH ofbetween 5.0 and 8.0 to produce a reduced glutathione solution having apredetermined initial concentration, wherein the concentration ofreduced glutathione decays over time in a linear fashion; b) preventingthe rate of reduced glutathione decay from exceeding 5% of thepredetermined initial concentration per month by storing the reducedglutathione solution at a predetermined temperature which is above thefreezing point of the solution and below 15° C., so as to maintain.